New β-alanine derivatives are orally available glucagon receptor antagonists

Article abstract of DOI:10.1021/jm058026u

A weak human glucagon receptor antagonist with an IC₅₀ of 7 μM was initially found by screening of libraries originally targeted to mimic the binding of the glucagon-like peptide (GLP-1) hormone to its receptor. Optimization of this hit for binding affinity for the glucagon receptor led to ligands with affinity in the nanomolar range. In addition to receptor binding, optimization efforts were made to stabilize the molecules against fast metabolic turnover. A potent antagonist of the human human glucagon receptor was obtained that had 17% oral availability in rats with a plasma half-life of 90 min. The major metabolites of this lead were identified and used to further optimize this series with respect to pharmacokinetic properties. This final optimization led to a potent glucagon antagonist that was orally available in rats and dogs and was efficacious in lowering blood glucose levels in a diabetic animal model.

Full text of DOI:10.1021/jm058026u

J. Med. Chem. 2007, 50, 113-128
113
New â-Alanine Derivatives Are Orally Available Glucagon Receptor Antagonists
Jesper Lau,*,† Carsten Behrens,† Ulla G. Sidelmann,† Lotte B. Knudsen,‡ Behrend Lundt,§ Christian Sams,§ Lars Ynddal,#
Christian L. Brand,| Lone Pridal, Anthony Ling,b Dan Kiel,∞ Michael Plewe,b Shengua Shi,× and Peter Madsen[
Protein Engineering, DiscoVery Biology Management, Medicinal Chemistry, Insulin Engineering, Drug Metabolism, Pharmacology Research,
and Screening & Assay Technology, NoVo Nordisk A/S, NoVo Nordisk Park, DK-2760 MåløV, Denmark, and Departments of Medicinal
Chemistry, Computational Chemistry, and Pharmacology, Pfizer Global Research and DeVelopment, 10770 Science Center DriVe,
San Diego, California 92121
ReceiVed May 3, 2005
A weak human glucagon receptor antagonist with an IC50 of 7 µM was initially found by screening of
libraries originally targeted to mimic the binding of the glucagon-like peptide (GLP-1) hormone to its receptor.
Optimization of this hit for binding affinity for the glucagon receptor led to ligands with affinity in the
nanomolar range. In addition to receptor binding, optimization efforts were made to stabilize the molecules
against fast metabolic turnover. A potent antagonist of the human human glucagon receptor was obtained
that had 17% oral availability in rats with a plasma half-life of 90 min. The major metabolites of this lead
were identified and used to further optimize this series with respect to pharmacokinetic properties. This
final optimization led to a potent glucagon antagonist that was orally available in rats and dogs and was
efficacious in lowering blood glucose levels in a diabetic animal model.
Introduction
resulted in an acute lowering of blood glucose, while in diabetic
ob/ob mice treated for 14 days a reduction in glycosylated
hemoglobin A1c (HbA1c) and triglyceride levels was observed.10
Thus, it has clearly been demonstrated that by preventing the
actions of glucagon in diabetic animals, it is possible to alleviate
their hyperglycemia. One glucagon antagonist is reported to be
effective and safe in human volunteers. During a hyperglu-
cagonemic period the effect of glucagon on blood glucose
concentration and rate of hepatic glucose production was blunted
in the group dosed with (+)-3,5-diisopropyl-2-(1-hydroxyethyl)-
6-propyl-4′-flouro-1,1′-biphenyl (Bay 27-9955).11
Glucagon, a 29 amino acid hormone, is the counter-regulatory
hormone to insulin such that when blood glucose levels are low,
the pancreatic R-cells secrete glucagon, which acts to promote
glucose production from the liver by stimulating both glyco-
genolysis (breakdown of glycogen) and gluconeogenesis (the
de novo synthesis of glucose from metabolic precursors). One
of the defects in type 2 diabetes is the apparent R-cell
“blindness” to elevated blood glucose, resulting in glucagon
secretion despite relatively high blood glucose and insulin levels.
This relative hyperglucagonemia leads to increased hepatic
glucose output and contributes to the insulin resistance that is
central to the etiology of diabetes.1-6 Human glucagon receptor
antagonists are thus expected to lower blood glucose by
alleviating one of the pathophysiological condition of type 2
diabetes, namely, excessive, uncontrolled glucagon action.
Furthermore, since this relative hyperglucagonemia is observed
during all stages of the disease, from the prediabetic impaired
glucose tolerance stage to late-stage type 2 diabetes, human
glucagon receptor (hGluR) antagonists are a new class of
compounds that could offer benefit to type 2 diabetic patients.
Non-peptide hGluR antagonists have been published, prima-
rily by scientists from the industry.12-30
The synthesis and biological activities from structure-
function studies with a focus on optimizing binding affinity for
the hGluR and the drug metabolism and pharmacokinetics
(DMPK) properties in parallel are reported here. This led to
the identification of potent hGluR antagonists with IC50 values
in the nanomolar range that were orally available and efficacious
in lowering blood glucose levels in diabetic animals.
Chemistry
Preclinical proof of concept comes from a number of
published studies demonstrating the blood glucose lowering
effects of glucagon immunoneutralizing monoclonal antibodies
when administered to diabetic rats,7,8 rabbits,9 and mice.10
Immunoneutralization of glucagon in these diabetic animals
Most compounds were prepared as single entities in parallel
libraries using either solid-phase or solution-phase synthesis
protocols. The initial libraries were prepared (method A) on a
polystyrene Wang resin (Scheme 1). An Fmoc protected amino
acid such as 3-aminopropionic acid 1 was attached to the resin
using symmetric anhydride mediated esterification followed by
deprotection. Subsequently p-bromomethylbenzoic acid was
coupled to the resin-bound â-alanine using standard peptide
chemistry. Introduction of the first variable proximal R1 group
was obtained by nucleophilic substitution with a variety of
primary amines. The reaction sequence was continued by
acylating the secondary amine with a set of carboxylic acids 6
in order to introduce the second set of distal R2 groups into the
library. This method was used for either semiautomated or fully
automated synthesis of single compounds in library format and
subsequently screened for binding to the human glucagon
receptor. An example of a library produced using the described
* To whom correspondence should be addressed. Phone: +4544434872.
Fax: +4544663450. E-mail: jela@novonordisk.com.
† Protein Engineering, Novo Nordisk A/S.
‡ Discovery Biology Management, Novo Nordisk A/S.
§ Medicinal Chemistry, Novo Nordisk A/S.
# Drug Metabolism, Novo Nordisk A/S.
| Pharmacology Research, Novo Nordisk A/S.
Screening & Assay Technology, Novo Nordisk A/S.
b Department of Medicinal Chemistry, Pfizer Global Research and
Development.
∞ Department of Pharmacology, Pfizer Global Research and Develop-
ment.
× Department of Computational Chemistry, Pfizer Global Research and
Development.
[ Insulin Engineering, Novo Nordisk A/S.
10.1021/jm058026u CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/04/2007

114 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Lau et al.
Figure 1. Design of exploration library.
Scheme 1. Preparation Method A
Scheme 2. Preparation Method B
solid-phase synthesis applying other amino acids is shown in
Figure 1. In general, only selected compounds that showed
significant binding affinity to hGluR were purified and char-
acterized in more details.
reductively alkylated with a set of aldehydes or ketones. The
reaction sequence was finalized by reaction of the secondary
amine 5 with a phenyl isocyanate 8, and finally the compounds
were cleaved from the resin.
An alternative protocol was developed in order to explore
the importance of the proximal R1 groups while keeping the
distal R3 group constant with urea connectivity (method B,
Scheme 2). In this procedure intermediate primary amine 7 was
On the basis of the initial libraries that were prepared and
screened for hGluR binding, it was evident that tert-butylcy-
cloalkyl was an important proximal substituent in the urea series.
It was accordingly decided to develop a general solution-phase

â-Analine DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 115
Scheme 3. Preparation Method C
Scheme 4. Preparation Method D
synthesis method diverting from key intermediate 16 with trans
stereochemistry, thereby allowing for preparation of larger
batches of interesting compounds to be used for further
biological characterization (Schemes 3 and 4). First, an attempt
was made to adjust the solid-phase procedure (method A) to a
solution-phase protocol (method C, Scheme 3). 4-Bromometh-
ylbenzoic acid methyl ester 9 was reacted with a cis/trans
mixture of 4-tert-butylcyclohexylamine 10. The formed mixture
of cis and trans isomers of 4-[(4-tert-butylcyclohexylamino)-
methyl]benzoic acid methyl ester (11 and 12) was separated by
chromatography on silica gel. These two isomers were used to
prepare the final trans isomer 25 as well as the corresponding
cis isomer 26. Though the cis and trans isomers had similar
binding affinity to hGluR, it turned out that the trans isomer
was metabolically more stable, and it was thus decided to
improve the synthesis of this particular isomer (method D,
Scheme 4). The advantage of this method was that intermediate
trans isomer of the imine 4-[(4-tert-butylcyclohexylimino)-
methyl]benzoic acid methyl crystallized out of the solution and
could be isolated by simple filtration. Subsequently it was
reduced to give trans-4-[(4-tert-butylcyclohexylamino)methyl]-
benzoic acid methyl ester 11. The remaining transformations
were simple functional group chemistries applicable for the
synthesis of a range of compounds to be used for generating
SAR information at the distal aryl group. Thus, 16 was reacted
with a variety of aryl isocyanates 17, and after final saponifica-
tion, a series of potential hGluR antagonists (25-38 and 58-
65) were obtained.
In order to scale up compounds having diverse proximal and
distal groups, a modified and efficient solution-phase method
(method E, Scheme 5) was developed in which â-alanine methyl
ester 19 was coupled to 4-formylbenzoic acid 20 to give the
key intermediate aldehyde 21. The aldehyde was reductively
aminated with primary amines and subsequently condensed with
isocyanates 8 and deprotected to give the final hGluR antagonists
in only four steps.
Biological Methods
The biological methods used are described in detail in the
Supporting Information.
Receptor assays were carried out using plasma membranes
from BHK cells expressing the cloned human glucagon receptor
as earlier described.19 Metabolic rates were estimated from
incubations with rat liver microsomes in order to rank com-
pounds in terms of their metabolic stability. Analysis was
performed by means of LC-MS. Selected compounds were
profiled with respect to the major metabolites formed in rat liver
microsomes. Metabolic profiles were analyzed by means of
LC-NMR and LC-MS-MS. The acute in vivo efficacy of
the compounds was studied in a glucagon challenged rat model
and in db/db mouse.

116 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Lau et al.
Figure 2. HTS hit and structure-activity overview.
Figure 3. Major metabolites of compound 25.
Scheme 5. Preparation Method E
Results and Discussion
target the hGluR in the design of new libraries. On the basis of
the structure of the initial hit, a series of followup libraries were
designed. The initial hit was considered potentially toxic because
of the 2-chloropyridyl moiety. In order to obtain initial SAR
information and to replace the undesirable 2-chloropyridyl
moiety, two initial libraries (96 and 384 members) were
produced. In the first 96-membered library the biphenylpropyl
of the original hit was replaced with 12 other lipophilic groups
A weak hGluR antagonist (Figure 2) with an IC50 of 7 µM
was found through high-throughput screening (HTS) of com-
pound libraries prepared on solid support and biased toward
unpublished hypotheses of the binding structure of the glucagon-
like peptide (GLP-1) hormone to its receptor. This initial hit
turned out to be a ligand for the GLP-1 receptor (IC50 ) 100
µM) as well as for the hGluR. It was accordingly decided to

â-Analine DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 117
and the 2-chloropyridyl was replaced with 8 other aromatic
groups. Screening of this library showed that the 2-chlorpyridyl
group could be replaced with several other aromatic groups,
such as benzothiophenes, to give hits with affinity in the
micromolar range. It was also found that the large lipophilic
biphenylpropyl group could be replaced with smaller moieties
such as 4-tert-butylbenzyl. In order to obtain ligands with
affinity in the nanomolar range for the hGluR, a second-
generation 384-membered library was designed (Figure 1). This
library was designed to explore the importance of the â-alanine
and the core benzoyl of the original hit.
Several potent hits were found in this library, and the first
indications of structural elements important for affinity to hGluR
were obtained. The aliphatic tert-butylcyclohexyl moiety was
included in several hits (R1 group in Figure 1). Because of the
smaller size and lower lipophilicity compared to the diphenyl-
propyl group in the initial hit, this group was among the selected
amines to be used in the further optimization. The preliminary
conclusions were based on screening of crude libraries, and it
was decided to include purified single compounds in the future
efforts to obtain a reliable SAR (Tables 1-4). Consideration
of the metabolic properties of this series was included in the
optimization that guided us to the lead structure 25 (Table 1)
that was shown to be orally available in dogs and to decrease
blood glucose levels in diabetic animals. In total, 4 libraries
(1056 compounds) were produced to find the lead structure 25.
Refinements of this structure with focus on the pharmacokinetics
(PK) and metabolic properties ultimately resulted in compound
57 (Table 3), which had attractive pharmacodynamic and
pharmacokinetic properties (Figures 5 and 6 and Table 5).
were found in the libraries where the phenyl group was
substituted with small electron-donating groups such as OMe,
OH, and aminoalkyl. Also, compounds that contained larger
electron-withdrawing substituents with heteroatoms in the
proximity of the phenyl group like CON(Et)2 in 31 had weaker
binding properties compared to the unsubstitued analogue 35.
Larger lipophilic substituents in the 4-position, e.g., 4-isopropyl
in 29 or 4-OCH2-cyclopropyl in 32, had a positive effect on
the binding properties when compared to the unsubstituted
analogue 35.
Compounds with two substituents in the phenyl group (R2
in Table 1) were also prepared. The SAR of these compounds
was more complex, and more compounds are needed to make
general conclusions. There are, however, examples with high
affinity among the disubstituted compounds such as 30, 37, and
38.
On the basis of these observations and the considerable data
obtained from the screening of crude libraries, it was concluded
that compounds containing either small electron-withdrawing
groups or larger lipophilic groups in the para position of the
distal phenyl moiety gave very potent antagonists with affinity
below 100 nM (Figure 2).
The nature of the proximal urea constituents was also
investigated. As exemplified with compounds 25 and 26, the
cis and trans isomers of the tert-butylcyclohexyl were equipo-
tent, and the overall structure-activity relationship of the series
was not markably influenced by the cis/trans geometry of this
group. The equipotency of the cis and trans isomers may be
explained by the conformational preferences of the two isomeric
compounds. In the cis isomer the chair conformation of the
cyclohexane ring is destabilized, shifting the equilibrium toward
a twist boat conformation, and the latter can easily be
superimposed on the chair conformation of the trans isomer.
One of the initial libraries was biased to â-alanine analogues
containing two lipophilic moieties (a distal benzothiazole and
a proximal aromatic or aliphatic group) at 8-11 Å distance from
an acidic group connected through a tertiary amide backbone.
One of the hits obtained 24, was resynthesized, and found to
be the most potent hit within this series of amide libraries.
On the basis of this promising result, it was decided to enlarge
the structural scope of this new class of hGluR antagonists. Both
solid support libraries and solution-phase single-compound
approaches were included in a structure-activity guided opti-
mization.
The importance of the 4-substituent of the cyclohexyl moiety
was investigated further. When tert-butyl was exchanged with
isopropyl as in 46, the binding affinity was not significantly
changed, whereas compounds with n-propyl in 47 were slightly
less potent. However, substituents containing heteroatoms like
CONH-alkyl in 40 and 41, COOH in 42, or OMe in 43 led to
inactive compounds. Derivatives with CON(alkyl)2 as in 44 or
OPh in 48 gave only weak ligands compared to the lead 25.
In order to optimize the geometrical spacing of the binding
moieties, it was decided to change the central amide connectivity
to the urea functionality. From the first urea-based libraries, it
became evident that compounds with 4-tert-butylcyclohexyl as
a proximal substituent consistently proved to give high-affinity
ligands when screened against the hGluR. Tables 1-4 show
the binding properties of hGluR antagonists that were selected
and purified from a series of libraries in order to obtain
information of the pharmacophores necessary for binding.
In Table 1, the influence of the substitution pattern of the
distal phenyl moiety on the binding to hGluR is shown. On the
basis of the series of trans isomers, the following structure-
activity relationship was observed: The electronic properties
of the substituents had a significant influence on the binding
properties. Selected small electron-withdrawing groups such as
4-OCF3 in 25, 3,5-di-CF3 in 30, 3-CN and 4-OCF3 in 37, and
3,4-di-Cl in 38 gave compounds with IC50 below 100 nM,
whereas the unsubstituted compound 35 had binding affinity
of around 1 µM. However, certain small electron withdrawing
groups do not provide high affinity, as the 4-CN substituent in
27 did not increase the binding affinity when compared to the
unsubstituted compound 35. In contrast to the effect observed
with selected small electron-withdrawing substituents, no hits
Among the various alternatives to the 4-(tert-butyl)cyclohexyl
moiety that were investigated, most were found to give inactive
compounds. One exception is using 4-cyclohexylphenyl instead
of 4-(tert-butyl)cyclohexyl as the proximal liphophilic moiety
exemplified in 56, which has similar binding affinity as the lead
25. In vitro metabolic studies gave important structural insight
on how to combine high in vitro affinity with acceptable in
vivo pharmacokinetic and dynamic properties. Incubation of
compounds containing the 4-cyclohexylphenyl in rat liver
microsomes showed that this moiety in general was metabolized
to the unsaturated 4-cyclohex-1-enylphenyl as the major me-
tabolite. This inspired us to introduce the 4-cyclohex-1-
enylphenyl, thereby eliminating a metabolic liability while
retaining affinity equivalent to that of the similar 4-cyclohexy-
lphenyl containing derivatives. Among the compounds synthe-
sized, the 3,5-dichloro derivative 57 was found to be an optimal
compound combining high receptor affinity with an acceptable
pharmacokinetics profile. The overall conclusion was that the
proximal part of the molecule had to be bulky and lipophilic
and that the size and shape were both crucial in order to obtain
highly active compounds (Figure 2).
The importance of the â-alanine moiety was also investigated
(Table 4). The significance of an acidic group was investigated

118 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Lau et al.
Table 1

â-Analine DeriVatiVes
Table 1 (Continued)
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 119
by preparing the corresponding primary amide 63 and the methyl
ether 59, which were both inactive. The GABA analogue 60
was also inactive, whereas the glycine analogue 62 and the
corresponding hydroxamic acid 65 had weak binding affinity
to the hGluR.
tertiary amines turned out to give active ligands but with weaker
binding affinity compared to 25. On the basis of these
observations, it was hypothesized that the urea moiety does not
directly have contact with the receptor cavity, but it is more
considered as a scaffold that keeps the important binding
moieties together in a geometry that is important for the binding
of the ligand to the receptor. The summary of the obtained SAR
is depicted in Figure 2. Further studies on the role of the central
urea core will be published separately. The original hit had a
weak affinity toward the GLP-1 receptor, and it was accordingly
interesting to test the selectivity of the optimized compounds.
25 and 57 were tested for GLP-1 receptor affinity in a GLP-1
binding assay. Both compounds had weak binding affinity to
the GLP-1 receptor (IC50 ) 1 and 4.9 µM, respectively), giving
a selectivity of 180-fold for 57 in favor of GluR.
Aiming for more active compounds, a set of constrained
analogues of 25 were prepared. The nipecotic acid analogue
61 had weak binding affinity, and the geminal dimethyl
substitued â-alanine 64 was inactive. On the basis of these
limited data, it is not obvious whether it is geometry or the
substitution of the secondary amide that caused the decrease in
binding affinity of 61. In conclusion, the acidic properties and
the geometry and position of this group were found to be very
crucial for binding of this series of hGluR antagonists.
The original hit 24 contained a tertiary amide core moiety,
but several attempts to optimize such compounds have so far
not resulted in more potent compounds compared to the
corresponding urea series. Replacement of the urea moiety with
Drug Metabolism and Pharmacokinetics. As mentioned
above, the in vitro metabolism was used together with the
receptor affinity studies to give combined structural information

120 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Lau et al.
Table 2

â-Analine DeriVatiVes
Table 2 (Continued)
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 121
on how to optimize this series of glucagon receptor antagonists
to orally available drug candidates. Whereas the initial hits had
very low oral availability and short plasma half-lives in rats,
the lead 25 was the first compound tested to have some oral
availability in rats and dogs (Table 5). The oral availability in
rats was found to be 17%, and the in vivo t1/2 value (after iv
dosing) was 90 min. The pharmacokinetic parameters in the
dog were similar (Fpo ) 10-20%, t1/2 ) 101 min (iv)). The
compound had a small volume of distribution in both rats and
dogs, and the half-life in plasma was accordingly expected to
be determined by the metabolic stability of the molecule.
Metabolite profiling of 25 was performed by LC-NMR and
LC-MS after incubation in rat liver microsomes in order to
unambiguously identify the major metabolites. The main
metabolism for 25 as well as for other similar compounds tested
was oxidation of the proximal tert-butylcyclohexyl moiety. As
mentioned earlier, these studies also revealed that compounds
containing a 4-cyclohexylphenyl moiety were oxidized to
4-cyclohexenylphenyl as the major metabolite.
observed for the compounds with the higher metabolic rates
(i.e., 25 gave a half-life of 90 min and oral availability of 17%,
whereas 29 gave a half-life of 20 min and only 2% oral
availability).
The plasma half-lives and the oral availabilities for the
cyclohexylphenyl and cylohexenylphenyl derivatives 56 and 57
were the same order of magnitude, and the oral bioavailabilities
were improved compared to 25. 57 was shown to be the
compound with the highest oral availability within this series,
and this compound was also found to have acceptable pharma-
cokinetic properties in dogs (Fpo ) 65%, t1/2 ) 92 min).
Pharmacodynamics. To demonstrate in vivo efficacy, the
lead compound 25 and the optimized derivative 57 were tested
in the glucagon challenged rat model in a dose-response dosing
regime. Both compounds inhibited the glucagon-stimulated rise
in blood glucose in a dose-dependent manner (Figure 5). In this
model 25 at a dose of 3 mg/kg iv significantly reduced blood
glucose caused by exogenous administration of glucagon. The
optimized compound 57 only showed significant effect at doses
above 3 mg/kg iv. Even though 57 has a higher affinity to hGluR
than 25, it had reduced in vivo potency relative to 25 in this rat
model. This may partly be explained by the species differences.
25 had similar affinity to the rat and human glucagon receptors
(IC50 ) 56 and 55 nM, respectively), whereas 57 had lower
affinity to the rat receptor compared to the human glucagon
receptor (IC50 ) 122 and 27 nM respectively).
The metabolic studies showed that the rate of metabolism
could be controlled by proper substitution of the distal aryl group
and the effect on metabolic stability of the distal substituents
as shown in Figure 4. The rate of metabolism of 29 having a
4-isopropyl substituent was much faster than the metabolism
of, for example, 25 and 37 that both contain a 4-OCF3
substituent. Structural investigations of the metabolism revealed
that in some of the compounds metabolism took place not only
in the proximal tert-butylcyclohexyl moiety but also in the distal
aryl moiety, which was the case for, 29 containing an isopropyl
group on the distal phenyl. The metabolic rates were reflected
in the in vivo PK experiments where short half-lives were
The conclusion from these acute glucagon-challenged studies
in rats was that both 25 and 57 are able to inhibit the rise in
blood glucose levels elicited by exogenous administered glu-
cagon, most likely because of the direct inhibition of glucagon
stimulated hepatic glucose output.

122 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Lau et al.
Table 3
The leptin-deficient obese model of type 2 diabetes, the ob/
ob mouse, was selected for further studies of 57. Predose blood
glucose levels were 17.1 ( 1.8 (n ) 6) and 18.3 ( 0.9 mM (n
) 6) in the vehicle and 57-treated mice, respectively. In vehicle-
treated mice, blood glucose levels changed by 0.9 ( 1.4, -0.2
( 2.2, -2.0 ( 1.8, and -1.9 ( 2.0 mM at 2, 4, 6, and 24 h
postdose, whereas blood glucose in the 57-treated mice changed
by -6.8 ( 1.0 (p ) 0.001), -8.4 ( 1.4 (p ) 0.010), -9.5 (
1.7 (p ) 0.012), and -1.8 ( 1.6 mM (p ) 0.964) at the
corresponding time points (Figure 6). Cumulative food intake
from 6 to 24 h postdose was 10.2 and 8.4 g/mouse in the vehicle
and 57-treated groups, respectively. From this study it is
concluded that 57 reduced the hyperglycemia for at least 6 h
after dosing with a maximum blood glucose reduction of 9.5
mM (Figure 6).
contain a carboxylic acid together with a proximal and a distal
lipophilic moiety positioned in a well-defined geometric orienta-
tion. A very steep SAR was demonstrated around the â-alanine
moiety, and it was crucial for potency that the proximal
lipophilic moiety did not contain heteroatoms. The largest
tolerance for structural variation was observed around the distal
aromatic group, where optimal binding was obtained when
selected electron-withdrawing substituents were attached. The
metabolic turnover was optimized in parallel to the binding
affinity in order to obtain compounds with higher possibilities
for appropriate pharmacokinetics and duration of action in whole
animals. 25 was shown to have an optimal lead profile for a
hGluR antagonist, and further in vivo studies confirmed this.
Further optimization of this series of compounds guided by in
vitro metabolism and structural characterization of metabolites
was crucial, leading to the cyclohexylphenyl and cyclohex-
enylphenyl derivatives 56 and 57 with high in vitro hGluR
binding affinity and acceptable PK properties. Furthermore, it
was demonstrated that 57 lowered blood glucose in a murine
type 2 diabetes model.
Conclusion
On the basis of the extensive examination of the SAR of this
new series of hGluR antagonists, it was concluded that it is
crucial for receptor recognition that the compounds of this series

â-Analine DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 123
Table 4
The resin-bound intermediate was successively washed with 3 ×
20 mL of THF, 3 × 20 mL of DMF, and 3 × 20 mL of MeOH.
The resin was dried in vacuo at 50 °C for 16 h to afford 3.77 g of
resin-bound 3-[4-(bromomethyl)benzoyl]aminopropionic acid.
Experimental Section
The preparation methods are illustrated by single representative
experimental procedures. Further experimental details can be found
in the patent literature.32
The above resin-bound 3-[4-(bromomethyl)benzoyl]aminopro-
pionic acid (1.0 g, 0.64 mmol) was treated with 2,2-diphenylethy-
lamine (1.26 g, 6.4 mmol) in 4 mL of DMSO. The mixture was
stirred at 80 °C for 12 h. Excess of reagents was removed by
filtration. The resin was successively washed with 3 × 10 mL of
DMSO and 3 × 10 mL of MeOH and dried in vacuo at 50 °C for
16 h to afford 1.07 g of resin-bound 3-(4-{[(N-{2,2-diphenylethyl}-
amino)methyl]benzoyl}amino)propionic acid.
The above resin-bound 3-(4-{[(N-{2,2-diphenylethyl}amino)-
methyl]benzoyl}amino)propionic acid (1.02 g, 0.61 mmol) was
suspended in THF and successively washed with 2 × 10 mL of
THF, 2 × 10 mL of 5% DIPEA in THF, and 5 × 10 mL of THF.
The resin slurry was then treated with with 5-chlorobenzo[b]-
thiophen-3-carboxylic acid (0.51 g, 2.4 mmol) in 4 mL of THF, 4
mL of pyridine, DIC (0.19 mL, 1.2 mmol), and 4-dimethylami-
nopyridine (24 mg, 0.12 mmol). The reaction mixture was stirred
at 25 °C for 12 h. The resin was drained and successively washed
with 3 × 10 mL of THF, 3 × 10 mL of DMF, and 5 × 10 mL of
DCM.
Method A: 3-(4-{[N-(5-Chlorobenzo[b]thiophen-3-carbonyl)-
N-(2,2-diphenylethyl)amino]methyl}benzoylamino)propionic Acid
24. Polystyrene resin derived with the Wang linker (1.07 mmol/g,
25.0 g, 26.75 mmol) was treated overnight at 25 °C with a solution
of N-Fmoc-3-aminopropionic acid (33.3 g, 107 mmol) activated
with DIC (8.5 mL, 54 mmol) in the presence of 4-dimethylami-
nopyridine (0.2 g) in 100 mL of THF. Excess of reagents was
removed by filtration. The resin-bound intermediate was succes-
sively washed with 3 × 100 mL of THF, 3 × 100 mL of DMF,
and 3 × 100 mL of MeOH. The resin was dried in vacuo at 50 °C
for 16 h to afford 31.54 g of resin-bound Fmoc-3-aminopropionic
acid.
The above resin-bound Fmoc-3-aminopropionic acid (3.95 g, 2.4
mmol) was treated with 40 mL of 50% piperidine in DMF for 15
min. The reagent was removed by filtration. The resin was
successively washed with 3 × 20 mL of DMF and 20 mL of a 1
M solution of HOBt in DMF. The resulting resin-bound intermedi-
ate was treated with a solution of 4-bromomethylbenzoic acid (2.15
g, 10 mmol) and HOBt (1.52 g, 10 mmol) in 25 mL of THF
activated by DIC (1.57 mL, 10 mmol). The reaction was performed
at 25 °C for 12 h. Excess of reagents was removed by filtration.
The resin-bound 3-(4-{[N-(5-chlorobenzo[b]thiophen-3-carbo-
nyl)-N-(2,2-diphenylethyl)amino]methyl}benzoylamino)propionic

124 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Lau et al.
Figure 6. Dynamic effect of compound 57 (100 mg/kg po) in ob/ob
mice.
Table 5. Pharmacokinetic Properties of Compounds 25, 29, 56, and 57
in Rats Dosed with 4 mg/kg po and 2 mg/kg iv
Cmax
Tmax
AUC
t1/2
Fpo
compound (ng mL-1
)
(min) (ng min-1 mL-1
)
(min, iv) (%)
25
29
56
57
106
40
316
275
60
15
240
60
13045
2597
95764
178226
90
20
64
82
17
2
54
69
Figure 4. Metabolic turnover of compounds in rat liver microsomes.
fractions were pooled and evaporated in Vacuo to afford 16.7 mg
of 3-(4-{[N-(5-chlorobenzo[b]thiophen-3-carbonyl)-N-(2,2-diphen-
ylethyl)amino]methyl}benzoylamino)propionic acid 24. Anal.
(C34H29ClN2O4S‚1.5H2O) C, H, N.
Method B: 3-{4-[1-(4-Propylcyclohexyl)-3-(4-trifluorometh-
oxyphenyl)ureidomethyl]benzoylamino}propionic Acid 47.
N-Fmoc-3-aminopropionic acid (47 mg, 150 µmol) was dissolved
in a mixture of 250 µL of DCM, 250 µL of DMF, and 100 µL of
DIPEA and added to 50 mg of polystyrene resin functionalized
with a 2-chlorotrityl chloride linker. After the suspension was
shaken for 4 h at 25 °C, the resin was isolated by filtration and
washed with 2 × 1 mL of DCM/MeOH/DIPEA 17:2:1 and 2 × 1
mL of DMF.
To the above resin-bound N-Fmoc-3-aminopropionic acid was
added 500 µL of a 20% solution of piperidine in DMF. Upon
shaking for 30 min, the resin was drained and washed with 1 mL
of DMF containing HOBt (50 mg/mL) and DMF (2 × 1 mL). Then
4-[(9H-fluoren-9-ylmethoxycarbonylamino)methyl]benzoic acid (74.2
mg, 200 µmol) dissolved in a mixture of 430 µL of DMF and 70
µL of diethylisopropylamine was added followed by bromo-tris-
pyrrolidinophosphonium hexafluorophosphate (PyBrOP, 93 mg, 200
µmol) dissolved in 500 µL of DMF. The mixture was shaken for
4 h at 25 °C followed by filtration and washing of the resin with
3 × 1 mL of DMF.
The Fmoc protecting group was removed from the above resin-
bound 3-{4-[(9H-fluoren-9-ylmethoxycarbonylamino)methyl]benz-
oylamino}propionic acid using 500 µL of a 20% solution of
piperidine in DMF. Upon shaking for 30 min, the resin was drained
and washed with 1 mL of a solution containing HOBt (50 mg/mL)
and DMF (2 × 1 mL), 2 × 1 mL of DCE, and 20 µL of acetic acid
dissolved in 1 mL of DCE.
The resulting resin-bound 3-(4-[aminomethyl]benzoylamino)-
propionic acid was treated with 4-propylcyclohexanone (98 mg,
700 µmol) dissolved in 500 µL of DCE, 50 µL of acetic acid, and
a slurry of NaBH(OAc)3 (148 mg, 700 µmol) in 1 mL of DCE.
Overnight shaking at 25 °C followed by filtration and washing with
2 × 1 mL of DCM, 2 × 1 mL of 1:1 CH3OH/DMF and 3 × 1 mL
of DMF afforded resin-bound 3-{4-[(4-propylcyclohexylamino)-
methyl]benzoylamino}propionic acid.
An amount of 200 µmol of 4-trifluoromethoxyphenyl isocyanate
dissolved in 500 µL of DCE was added to the above resin-bound
3-{4-[(4-propylcyclohexylamino)methyl]benzoylamino}propionic
acid. Shaking the mixture for 5 h at 25 °C followed by filtration
and washing of the resin with 2 × 1 mL of DCM, 4 × 1 mL of
Figure 5. Dynamic effect of compounds 25 and 57 in glucagon
challenged rats. Anesthetized animals were given an intravenous dose
of compounds 5 min prior to a 3 µg/kg glucagon load.
acid 24 was treated with a 50% solution of TFA in DCM (10 mL).
The cleavage mixture was stirred for 45 min at 25 °C. The resin
was drained and washed with DCM several times. The combined
filtrates were concentrated in vacuo. The residue was dissolved in
a 1:1 mixture of MeOH and DCM (1 mL) and concentrated in vacuo
to afford 0.359 g of crude 24. The crude product (50 mg) was
purified by column chromatography on RP-C18 silica gel (Sep-
Pak, Waters), eluting with a mixture of MeCN and water. Pure

â-Analine DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 125
DMF, 2 × 1 mL of H2O, 3 × 1 mL of THF, and 3 × 1 mL of
DCM afforded the resin-bound 47.
methyl]benzoylamino}propionic acid ethyl ester. Mp ) 108-111
°C. 1H NMR (DMSO-d6), 400 MHz: δ 8.55 (s, 1H), 8.50 (t, 1H),
7.75 (d, 2H), 7.55 (d, 2H), 7.31 (d, 2H), 7.22 (d, 2H), 4.60 (s, 2H),
4.10-4.00 (m, 1H), 3.60 (s, 3H), 3.45 (dd, 2H), 2.55 (t, 2H), 1.80-
1.60 (m, 4H), 1.50-0.80 (m, 5H), 0.80 (s, 9H).
The above resin-bound 47 was treated with 1 mL of 5% TFA in
DCM for 1 h at 25 °C. The product was filtered off, and the resin
was washed with 1 mL of DCM. The combined extracts were
concentrated in vacuo. The residue was dissolved in 50 µL of
DMSO + 500 µL of CH3CN and purified by preparative HPLC
using a Supelcosil ABZ+ 25 cm × 10 mm, 5 µm column. The
starting eluent composition was 5% CH3CN in H2O, changing over
30 min to 90% CH3CN in H2O, which was then kept constant for
5 min before going back to the starting composition over 10 min.
The flow rate was kept constant at 8 mL/min, collecting one fraction
per minute. The process was monitored using a UV detector op-
erating at 214 nm. The fractions containing the desired products
were combined and evaporated in vacuo to afford 3-{4-[1-(4-
propylcyclohexyl)-3-(4-trifluoromethoxyphenyl)ureidomethyl]benz-
oylamino}propionic acid 47. Anal. (C28H34 F3N3O5‚0.5H2O) C, H,
N.
trans-3-{4-[1-(4-tert-Butylcyclohexyl)-3-(4-trifluoromethoxy-
phenyl)ureidomethyl]benzoylamino}propionic acid ethyl ester (1.4
g, 2.4 mmol) was suspended in ethanol (50 mL), and NaOH (4 N,
6 mL) was added. The reaction mixture was stirred for 2 h at 50
°C and then concentrated in vacuo until all EtOH was removed.
The reaction mixture was diluted with water (100 mL) and adjusted
to pH 2 with 4 N HCl, and trans-3-{4-[1-(4-tert-butylcyclohexyl)-
3-(4-trifluoromethoxyphenyl)ureidomethyl]benzoylamino}propi-
1
onic acid 25 was isolated by filtration. Mp ) 152-154 °C. H
NMR (DMSO-d6), 400 MHz: δ 12.20 (s, 1H), 8.55 (s, 1H), 7.95
(t, 1H), 7.75 (d, 2H), 7.55 (d, 2H), 7.31 (d, 2H), 7.22 (d, 2H), 4.60
(s, 2H), 4.10-4.00 (m, 1H), 3.45 (dd, 2H), 2.50 (2H), 1.80-1.60
(m, 4H), 1.45-1.35 (m, 2H), 1.15-1.05 (m, 2H), 0.95-0.85 (m,
1H), 0.80 (s, 9H). Anal. (C29H36F3N3O5) C, H, N.
Method C: trans-3-{4-[1-(4-tert-Butylcyclohexyl)-3-(4-tri-
fluoromethoxyphenyl)ureidomethyl]benzoylamino}propionic Acid
25. 4-(Bromomethyl)benzoic acid methyl ester (5.0 g, 22 mmol)
and 4-tert-butylcyclohexylamine (as a 17/83% cis/trans mixture)
(3.4 g, 22 mmol) were dissolved in DMF, and K2CO3 (6.1 g, 44
mmol) was added. The reaction mixture was stirred at 100 °C for
7 h and for 16 h at 20 °C. Water (100 mL) and EtOAc (200 mL)
were added to the reaction mixture. The organic phase was isolated
and washed with water (2 × 100 mL) and brine (2 × 100 mL).
The organic phase was dried (MgSO4), filtered, and concentrated
in vacuo to give a cis/trans mixture of 4-[(4-tert-butylcyclohexy-
lamino)methyl]benzoic acid methyl ester as a crude product. The
two isomers were separated on silica (110 g) using a mixture of
EtOAc and DCM (7:3) as eluent.
Method D: trans-3-{4-[3-(3,5-Bistrifluoromethylphenyl)-1-
(4-tert-butylcyclohexyl)ureidomethyl]benzoylamino}propionic Acid
30. 4-Formylbenzoic acid methyl ester (10.6 g, 64.4 mmol) was
dissolved in MeOH (200 mL). A 17:83 cis/trans mixture of 4-tert-
butylcyclohexylamine (10.0 g, 64.4 mmol, Aldrich) was added,
leading to immediate precipitation of white crystals. The mixture
was heated to reflux for 30 min to complete imine formation and
cooled to 0 °C on an ice bath. The crystalline pure trans-4-[(4-
tert-butylcyclohexylimino)methyl]benzoic acid methyl ester was
then collected by filtration and dried overnight in vacuo to afford
1
15.3 g (78%). H NMR (CDCl3), 300 MHz: δ 8.37 (s, 1H), 8.06
(d, 2H), 7.77 (d, 2H), 3.92 (s, 3H), 3.17 (m, 1H), 1.83 (m, 4H),
1.60 (m, 2H), 1.09 (m, 3H), 0.87 (s, 9H). Anal. (C19H27NO2) C, H,
N.
Trans Isomer 11. Anal. (C19H29NO2) C, H, N. 1H NMR
(DMSO-d6), 400 MHz: δ 7.90 (d, 2H), 7.48 (d, 2H), 3.82 (s, 3H),
3.78 (s, 2H), 2.30-2.20 (m, 1H), 2.05-1.90 (m, 3H), 1.73-1.65
(m, 2H), 1.10-0.90 (m, 4H), 0.80 (s, 9H).
The mother liquor was evaporated to dryness in vacuo to leave
4.2 g (22%) of a white solid, which according to the NMR spectrum
1
consisted mainly of the imino cis isomer. H NMR (CDCl3), 300
Cis Isomer 12. 1H NMR (DMSO-d6), 400 MHz: δ 7.92 (d, 2H),
7.58 (d, 2H), 3.90 (dd, 1H), 3.85 (s, 3H), 3.80 (dd, 1H), 2.50-
2.35 (m, 1H), 2.00-1.85 (m, 2H), 1.80-1.70 (m, 2H), 1.70-1.45
(m, 2H), 1.00-0.80 (m, 1H), 0.80 (s, 9H).
MHz: δ 8.36 (s, 1H), 8.07 (d, 2H), 7.81 (d, 2H), 3.92 (s, 3H),
3.54 (m, 1H), 1.55-1.92 (m, 8H), 1.14 (m, 1H), 0.90 (s, 9H).
trans-4-[(4-tert-Butylcyclohexylimino)methyl]benzoic acid meth-
yl ester (21.0 g, 69.2 mmol) was suspended in MeOH (300 mL),
and acetic acid (50 mL) was added. To the resulting clear solution
was added NaCNBH3 (3.5 g, 55.5 mmol), and the mixture was
stirred at ambient temperature for 30 min. The reaction volume
was then reduced to one-third by rotary evaporation, and EtOAc
(500 mL) was added. The mixture was washed with sodium
carbonate solution (5%, 500 mL) and dried (Na2SO4). The solvent
was removed in vacuo to afford 21.1 g (100%) of 11 as a white
A solution of 12 (2.6 g, 8.6 mmol) and 4-(trifluoromethoxy)-
phenyl isocyanate (1.7 g, 8.6 mmol) in MeCN (40 mL) was stirred
at 20 °C for 16 h. The reaction mixture was concentrated in vacuo,
and the crude product was purified on silica (100 g) using heptane
and EtOAc (3:1) as eluent to give trans-4-[1-(4-tert-butylcyclo-
hexyl)-3-(4-trifluoromethoxyphenyl)ureidomethyl]benzoic acid meth-
yl ester. The product was suspended in EtOH (80 mL), and NaOH
(4 N, 17 mL) was added. The reaction mixture was stirred at 50
°C for 3 h and then concentrated in vacuo until all ethanol was
removed. The reaction mixture was diluted with water (100 mL)
and adjusted to pH 2 with 4 N HCl. The aqueous phase was
extracted with EtOAc (3 × 75 mL), and the combined organic
phases were dried (MgSO4) and concentrated in vacuo to give trans-
4-[1-(4-tert-butylcyclohexyl)-3-(4-trifluoromethoxyphenyl)ureidom-
1
crystalline solid sufficiently pure for further reactions. H NMR
(CDCl3), 300 MHz: δ 7.98 (d, 2H), 7.38 (d, 2H), 3.90 (s, 3H),
3.86 (s, 2H), 2.39 (m, 1H), 2.01 (m, 2H), 1.77 (m, 2H), 1.51 (bs,
1H), 0.93-1.18 (m, 5H), 0.82 (s, 9H).
Compound 11 (20.0 g, 65.9 mmol) was dissolved in THF (300
mL). Di-tert-butyl pyrocarbonate (16.0 g, 73.4 mmol) and DIPEA
(12.0 g, 92.9 mmol) were added, and the clear solution was stirred
overnight at ambient temperature. The solvent was removed in
vacuo, and crude 14 was redissolved in EtOH (200 mL), and
aqueous NaOH solution (100 mL, 4 N) was added whereafter the
mixture was heated to 70 °C for 4 h. After the mixture was cooled,
the reaction volume was reduced to one-third by rotary evaporation
and water (300 mL) was added. The mixture was extracted with
diethyl ether (2 × 200 mL) to remove traces of nonhydrolyzed
material. The aqueous phase was then acidified to pH 3.0 by
addition of aqueous 4 N HCl. The solid formed was isolated by
filtration, and the crystals were washed once with water and dried
overnight in vacuo (40 °C) to afford 24.3 g (93%) of trans-4-{-
[N-(tert-butoxycarbonyl)-N-(4-tert-butylcyclohexyl)amino]methyl}-
1
ethyl]benzoic acid. H NMR (DMSO-d6), 400 MHz: δ 12.80 (s,
1H), 8.55 (s, 1H), 7.90 (d, 2H), 7.55 (d, 2H), 7.35 (d, 2H), 7.21 (d,
2H), 4.62 (s, 2H), 4.10-4.00 (m, 1H), 2.00 (s, 2H), 1.80-1.60
(m, 4H), 1.48-1.38 (m, 2H), 1.20-1.00 (m, 2H), 1.00-0.88 (m,
1H), 0.80 (s, 9H).
trans-4-[1-(4-tert-Butylcyclohexyl)-3-(4-trifluoromethoxypheny-
l)ureidomethyl]benzoic acid (2.0 g, 4.1 mmol), HOBt (0.6 g, 4.3
mmol), and DIPEA (0.8 g, 4.3 mmol) were dissolved in DMF (40
mL). A solution of DIPEA (0.5 g, 4.1 mmol) and 3-aminopropionic
acid ethyl ester hydrochloride (0.4 g, 4.3 mmol) in DMF (10 mL)
was added, and the reaction mixture was stirred for 16 h at 20 °C.
EtOAc (150 mL) and water (100 mL) were added, and the organic
phase was isolated. The aqueous phase was extracted with EtOAc
(50 mL), and the organic phases were combined, dried, (MgSO4)
and concentrated in vacuo. The crude product was purified on silica
(80 g) using heptane and EtOAc (1:1) as eluent to give trans-3-
{4-[1-(4-tert-butylcyclohexyl)-3-(4-trifluoromethoxyphenyl)ureido-
1
benzoic acid 14. H NMR (CDCl3), 300 MHz: δ 8.04 (d, 2H),
7.31 (d, 2H), 4.39 (bs, 2H), 4.05 (bs, 1H), 1.78 (bd, 4H), 0.95-
1.65 (m, 14 H), 0.83 (s, 9H). The signals were broad because of
the presence of cis/trans carbamate isomers. Anal. (C23H35NO4) C,
H, N.

126 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Lau et al.
trans-4-{[N-(tert-Butoxycarbonyl)-N-(4-tert-butylcyclohexyl)-
amino]methyl}benzoic acid (22.0 g, 56.5 mmol) and HOBt (8.6 g,
57.0 mmol, containing 12% w/w water) were dissolved in DMF
(300 mL). N-Dimethylaminopropyl-N′-ethylcarbodiimide hydro-
chloride (10.9 g, 56.8 mmol), â-alanine methyl ester hydrochloride
(8.4 g, 60 mmol), and DIPEA (25 mL, 142 mmol) were added,
and the clear solution was stirred at 20 °C for 16 h. The solvent
was removed in vacuo, and the residual oil was redissolved in
EtOAc (500 mL). The organic phase was washed with 5% aqueous
sodium hydrogen carbonate solution (2 × 500 mL), 0.5 M citric
acid (2 × 250 mL), and brine before being dried (Na2SO4). The
solvent was evaporated in vacuo, and the residual oil was evaporated
twice from MeCN to afford 24.0 g (89%) of 15. 1H NMR (CDCl3),
300 MHz: δ 7.69 (d, 2H), 7.28 (d, 2H), 6.81 (t, 1H), 4.38 (bs,
2H), 3.23 (s, 3H), 3.21 (t, 2H), 2.66 (t, 2H), 1.75 (bd, 4H), 0.95-
1.65 (m, 14 H), 0.80 (s, 9H).
Intermediate 15 (19.5 g, 41.1 mmol) was dissolved in a mixture
of TFA (200 mL) and DCM (200 mL), while cooling in an ice
bath. The ice bath was removed, and the mixture was allowed to
stir at room temperature for 30 min. The solvent was evaporated
in vacuo, and the residue was crystallized from EtOAc/heptane to
afford 14.8 g (74%) of the trifluoroacetate of 16. 1H NMR (CDCl3),
300 MHz: δ 9.98 (trace of TFA), 8.06 (bs, 2H), 7.73 (d, 2H), 7.41
(d, 2H), 7.34 (t, 1H), 4.21 (t, 2H), 3.75 (s, 3H), 3.74 (t, 2H), 3.04
(m, 1H), 2.70 (t, 2H), 2.17 (bd, 2H), 1.95 (bd, 2H), 1.50 (m, 2H),
0.92-1.15 (m, 3H), 0.85 (s, 9H). Anal. (C22H34N2O3‚C2HF3O2) C,
H, N.
3,5-Bis(trifluoromethyl)aniline (2.0 g, 8.7 mmol) was dissolved
in DCM (80 mL), and N,N-DIPEA (1.6 mL, 9.6 mmol) was added
followed by slow addition of phenyl chloroformate (1.1 mL, 8.7
mmol). The resulting mixture was stirred at room temperature for
16 h. The mixture was successively washed with 1 N hydrochloric
acid (3 × 100 mL), water (3 × 100 mL), a mixture of water and
saturated aqueous sodium hydrogen carbonate (1:1, 2 × 100 mL),
and a mixture of water and brine (1:1, 3 × 100 mL), dried (MgSO4),
and concentrated in vacuo. The residue was crystallized from
heptane containing a little diethyl ether to afford 1.3 g (43%) of
(3,5-bistrifluoromethylphenyl)carbamic acid phenyl ester as a solid.
Anal. (C15H9F6NO2) C, H, N.
temperature for 16 h. The mixture was diluted with EtOAc (50
mL) and washed with a saturated aqueous solution of sodium
chloride (2 × 50 mL). Drying (MgSO4) and concentration afforded
2.23 g (75%) of 1-cyclopropylmethoxy-4-nitrobenzene as an oil.
1H NMR (CDCl3): δ 8.12 (2H, d), 6.94 (2H, d), 3.89 (2H, d), 1.30
(1H, m), 0.58 (2H, m), 0.40 (2H, m).
The above 1-cyclopropylmethoxy-4-nitrobenzene (2.0 g, 9.5
mmol) was dissolved in EtOH (50 mL), and tin(II) chloride
dihydrate (10.7 g, 48 mmol) was added. The mixture was refluxed
for 24 h. After cooling, the mixture was concentrated in vacuo.
The residue was dissolved in EtOAc (40 mL) and 1 N NaOH (180
mL). This mixture was filtered through Celite. The aqueous phase
was extracted with EtOAc (2 × 50 mL), and the combined organic
extracts were dried (MgSO4) and concentrated in vacuo to afford
1
0.88 g (52%) of 4-cyclopropylmethoxyaniline. H NMR (DMSO-
d6): δ 6.62 (2H, d), 6.4-6.5 (3H, m), 4.55 (2H, s), 3.63 (2H, d),
1.15 (1H, m), 0.53 (2H, m), 0.25 (2H, m).
32 was prepared from the above aniline and 16, similar to method
E for 30. 1H NMR (DMSO-d6): δ 7.76 (2H, d), 7.40 (2H, d), 7.10
(2H, d), 6.88 (1H, t), 6.77 (2H, d), 6.08 (1H, s), 4.52 (2H, s), 4.14
(1H, t), 2.70 (4H, m), 1.9 (4H, bt), 1.1-1.4 (5H, m), 0.92 (1H, m),
0.85 (9H, s), 0.62 (2H, m), 0.42 (2H, m).
trans-3-{4-[3-(2-Bromo-4-trifluoromethoxyphenyl)-1-(4-tert-
butylcyclohexyl)ureidomethyl]benzoylamino}propionic Acid 34.
4-Trifluoromethoxyaniline (1.0 g, 5.6 mmol) was dissolved in
HOAc (10 mL). Bromine (585 µL, 11 mmol) dissolved in HOAc
(2 mL) was added with stirring for 10 min at room temperature.
The resulting mixture was stirred at room temperature for 2 h and
then poured into water (100 mL). The solid formed (2,5-dibromo-
4-trifluoromethoxyaniline) was filtered off. The filtrate was made
alkaline with solid NaOH and extracted with DCM (100 mL), dried
(MgSO4), and concentrated (30 °C, 200 mbar) to afford 0.57 g
1
(40%) of 2-bromo-4-trifluoromethoxyaniline. H NMR (DMSO-
d6): δ 7.37 (1H, d), 7.10 (1H, dd), 6.85 (1H, d), 5.55 (2H, bs).
From this and 16, trans-3-{4-[3-(2-bromo-4-trifluoromethox-
yphenyl)-1-(4-tert-butylcyclohexyl)ureidomethyl]benzoylamino}-
1
propionic acid 34 was similarly prepared as described for 30. H
NMR (DMSO-d6): δ 8.26 (2H, d), 7.76 (2H, d), 7.43 (2H, d), 7.31
(1H, d), 7.16 (1H, dd), 6.88 (1H, s), 6.85 (1H, t), 4.60 (2H, s),
4.16 (1H, t), 3.75 (2H, q), 2.74 (2H, t), 1.9 (4H, m), 1.4 (2H, m),
1.2 (2H, m), 0.95 (1H, m), 0.86 (9H, s).
The above carbamic acid phenyl ester (1.0 g, 2.9 mmol) was
mixed with 16 (1.07 g, 2.9 mmol), prepared as described above,
and triethylamine (1.2 mL, 8.6 mmol) in DCM (55 mL). The
resulting mixture was refluxed for 4 h. The cooled reaction mixture
was diluted with toluene (50 mL) and washed with water (3 × 50
mL) followed by a mixture of water and saturated aqueous sodium
chloride (1:1, 3 × 100 mL), dried (MgSO4), and concentrated in
vacuo to afford 1.2 g (67%) of 3-{4-[3-(3,5-bistrifluoromethylphe-
nyl)-1-(4-tert-butylcyclohexyl)ureidomethyl]benzoylamino}propi-
trans-3-{4-[1-(4-tert-Butylcyclohexyl)-3-(4-trifluoromethoxy-
phenyl)ureidomethyl]benzoylamino}propionic Acid 25. Inter-
mediate 16 (10.0 g 24 mmol) was suspended in MeCN (300 mL),
and DIPEA (4.14 mL, 24 mmol) was added. To this suspension
4-trifluoromethoxyphenyl isocyanate (3.75 mL, 24 mmol) was
added. Stirring at room temperature was continued for 4 h, and
then the mixture was left at 5 °C for 16 h. Filtration and washing
with cold MeCN afforded 11.9 g (85%) of trans-3-{4-[1-(4-tert-
butylcyclohexyl)-3-(4-trifluoromethoxyphenyl)ureidomethyl]ben-
zoylamino}propionic acid methyl ester. Hydrolysis of this ester
afforded 11 g (94%) of trans-3-{4-[1-(4-tert-butylcyclohexyl)-3-
(4-trifluoromethoxyphenyl)ureidomethyl]benzoylamino}propion-
ic acid 25. Mp 152-154 °C. Anal. (C29H36F3N3O5) C, H, N.
General Procedure for Preparing Isocyanates from (Substi-
tuted) Anilines and Diphosgene. To a solution of the aniline in
anhydrous toluene was added dry 1 N HCl in diethyl ether (5 equiv).
A precipitate was formed. The toluene was evaporated. To the solid
was added more anhydrous toluene, and the toluene was evaporated
again to remove excess HCl. This procedure was repeated 2-3
times. To a mixture of the solid in toluene (about 10 g in 300 mL)
was added diphosgene (trichloromethyl chloroformate) (10 equiv
or more) or phosgene. The mixture was refluxed under nitrogen
overnight. A clear solution was obtained. If more solid was present,
more diphosgene was added and reflux continued. When a clear
solution was obtained, the reaction mixture was concentrated in
vacuo at 60-90 °C to remove the toluene and excess diphosgene
until the theoretical weight was obtained. To the crude isocyanate
was added more anhydrous toluene, and the mixture was concen-
trated again to remove excess HCl. The crude isocyanate was used
1
onic acid methyl ester as a solid. H NMR (CDCl3): δ 7.80 (s,
2H), 7.77 (d, 2H), 7.46 (s, 1H), 7.38 (d, 2H), 6.90 (t, 1H), 6.80 (s,
1H), 4.57 (s, 2H), 4.18 (bt, 1H), 3.72 (m, 5H), 7.62 (t, 1H), 1.88
(bt, 4H), 0.9-1.5 (m, 5H), 0.83 (s, 9H).
The above propionic acid methyl ester (1.0 g, 1.6 mmol) was
dissolved in warm EtOH (50 mL), and after the mixture was cooled
to room-temperature, 4 N NaOH (6 mL) was added. The reaction
mixture was stirred at room temperature for 1 h. Glacial acetic acid
(10 mL) was added, and the mixture was concentrated in vacuo.
The residue was partitioned between water (50 mL) and EtOAc (2
× 50 mL). The combined organic phases were dried (MgSO4) and
concentrated in vacuo to afford 0.88 g (93%) of trans-3-{4-[3-
(3,5-bistrifluoromethylphenyl)-1-(4-tert-butylcyclohexyl)ureidomethyl]-
benzoylamino}propionic acid 30. 1H NMR (DMSO-d6): δ 12 (bs,
1H), 9.00 (s, 1H), 8.45 (t, 1H), 8.24 (s, 2H), 7.60 (d, 2H), 7.32 (d,
2H), 4.62 (s, 2H), 4.05 (bt, 1H), 3.45 (q, below water, 2H), 2.50 (t,
below DMSO, 2H), 1.70 (bt, 4H), 0.9-1.5 (m, 5H), 0.83 (s, 9H).
Anal. (C30H35F6N3O4) C, H, N.
trans-3-{4-[1-(4-tert-Butylcyclohexyl)-3-(4-cyclopropylmeth-
oxyphenyl)ureidomethyl]benzoylamino}propionic Acid 32. 4-Ni-
trophenol (2.0 g, 14.4 mmol) was dissolved in DMF (50 mL).
K2CO3 (6.0 g, 43 mmol) and bromomethylcyclopropane (1.51 mL,
16 mmol) were added, and the resulting mixture was stirred at room

â-Analine DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 127
without further purification. This method gives the isocyanates in
pure form, which may be used in the next step.
methyl ester as an oil. 1H NMR (CDCl3): δ 2.70 (2H, t), 3.69 (3H,
s), 3.70 (2H, q), 7.68 (1H, bt), 7.9-8.0 (4H, m), 10.1 (1H, s).
The above compound 21 (2.0 g, 8.5 mmol) was dissolved in
DMF (20 mL). Triethyl orthoformate (10 mL), glacial acetic acid
(1 mL), NaCNBH3 (0.81 g, 12.8 mmol), and 4-tert-butylaniline
(1.27 g, 8.5 mmol) were added, and the resulting mixture was stirred
at room temperature for 16 h. To the mixture was added saturated
aqueous sodium chloride (100 mL), and the mixture was extracted
with EtOAc (3 × 100 mL). The combined organic extracts were
washed with saturated aqueous sodium chloride (3 × 100 mL).
The organic phase was dried (MgSO4) and concentrated in vacuo.
The residue was purified by column chromatography on silica gel,
eluting with a mixture of EtOAc and heptane (1:1) to afford 0.87
g (30%) of 3-{4-[(4-tert-butylphenylamino)methyl]benzoylamino}-
cis-3-{4-[1-(4-tert-Butylcyclohexyl)-3-(4-trifluoromethoxy-
phenyl)ureidomethyl]benzoylamino}propionic Acid 26. 4-[(cis-
4-tert-Butylcyclohexylamino)methyl]benzoic acid methyl ester (0.36
g, 1.2 mmol) and 4-(trifluoromethoxy)phenyl isocyanate (0.24 g,
1.2 mmol) were dissolved in MeCN (10 mL) and stirred at 20 °C
for 16 h. The reaction mixture was concentrated in vacuo, and the
crude product was purified on silica (25 g) using heptane and EtOAc
(9:1) as eluent to give cis-4-[1-(4-tert-butylcyclohexyl)-3-(4-trif-
luoromethoxyphenyl)ureidomethyl]benzoic acid methyl ester. The
product was suspended in EtOH (10 mL), and NaOH (4 N, 1.1
mL) was added. The reaction mixture was stirred at 50 °C for 3 h
and then concentrated in vacuo until all EtOH was removed. The
reaction mixture was diluted with water (50 mL) and adjusted to
pH 2 with hydrochloric acid (4 N). The aqueous phase was extracted
with EtOAc (75 mL), and the organic phase was dried (MgSO4)
and concentrated in vacuo to give cis-4-[1-(4-tert-butylcyclohexyl)-
3-(4-trifluoromethoxyphenyl)ureidomethyl]benzoic acid 26. 1H
NMR (DMSO-d6), 400 MHz: δ 12.80 (s, 1H), 8.61 (s, 1H), 7.90
(d, 2H), 7.55 (d, 2H), 7.35 (d, 2H), 7.22 (d, 2H), 4.72 (s, 2H),
4.32-4.22 (m, 1H), 1.85-1.70 (m, 2H), 1.65-1.45 (m, 4H), 1.40-
1.10 (m, 3H), 0.80 (s, 9H). Anal. (C26H31F3N2O4) C, H, N.
1
propionic acid methyl ester as an oil. H NMR (CDCl3): δ 7.73
(2H, d), 7.44 (2H, d), 7.19 (2H, d), 6.83 (2H, d), 6.57 (2H, d,)
4.38 (2H, s), 4.04 (1H, s), 3.73 (5H, m), 2.67 (2H, t), 1.28 (9H, s).
The above 3-{4-[(4-tert-butylphenylamino)methyl]benzoylamino}-
propionic acid methyl ester (0.82 g, 2.2 mmol) was dissolved in
MeCN (15 mL). N,N-DIPEA (378 µL, 2.2 mmol) and 4-(trifluo-
romethoxy)phenyl isocyanate (500 µL, 3.3 mmol) were added. The
resulting mixture was stirred at room temperature for 5 h and was
refluxed for 16 h. The cooled reaction mixture was concentrated
in vacuo, and the residue was dissolved in EtOAc (50 mL) and
washed with water (2 × 50 mL), dried (MgSO4), and concentrated
in vacuo. The residue was crystallized from a mixture of EtOAc
and heptane (1:1) containing 1% glacial acetic acid to afford 0.40
g (32%) of 3-{4-[1-(4-tert-butylphenyl)-3-(4-trifluoromethoxyphenyl)-
ureidomethyl]benzoylamino}propionic acid methyl ester as a solid.
1H NMR (CDCl3): δ 7.70 (2H, d), 7.43 (2H, d), 7.3-7.4 (4H, m),
7.1 (4H, m), 6.80 (1H, t), 6.30 (1H, s), 4.94 (2H, s), 3.72 (5H, m),
2.67 (2H, t), 1.34 (9H, s),
cis-4-[1-(4-tert-Butylcyclohexyl)-3-(4-trifluoromethoxyphenyl)-
ureidomethyl]benzoic acid (0.3 g, 0.6 mmol), HOBt (0.1 g, 0.7
mmol), and DIPEA (0.13 g, 0.7 mmol) were dissolved in DMF
(10 mL). DIPEA (0.1 g, 0.7 mmol) and 3-aminopropionic acid ethyl
ester hydrochloride (0.07 g, 0.7 mmol) in DMF was added, and
the reaction mixture was stirred for 16 h at 20 °C. EtOAc (80 mL)
and water (50 mL) were added, and the organic phase was isolated.
The aqueous phase was extracted with EtOAc (50 mL), and the
combined organic phases were dried (MgSO4) and concentrated in
vacuo. The residue was crystallized from heptane and EtOAc (4:
1) to give 3-{4-[1-(cis-4-tert-butylcyclohexyl)-3-(4-trifluoromethoxy-
phenyl)ureidomethyl]benzoylamino}propionic acid ethyl ester. Mp
The above propionic acid methyl ester (0.25 g, 0.44 mmol) was
dissolved in 1,4-dioxane (25 mL), and then 4 N NaOH (6 mL)
was added. The mixture was stirred at room temperature for 16 h,
and then 36% aqueous hydrochloric acid (10 mL) was added. The
mixture was extracted with EtOAc (3 × 50 mL). The combined
organic extracts were dried (MgSO4) and concentrated in vacuo.
The residue was crystallized from a mixture of diethyl ether and
heptane to afford 0.10 g (42%) of 3-{4-[1-(4-tert-butylphenyl)-3-
(4-trifluoromethoxyphenyl)ureidomethyl]benzoylamino}propion-
ic acid 50. Anal. (C29H30F3N3O5‚0.5H2O) C, H, N. 1H NMR
(CDCl3): δ 7.68 (2H, d), 7.43 (2H), 7.35 (2H, d), 7.28 (2H, d),
7.1 (4H, m), 6.88 (1H, t), 6.32 (1H, s), 4.95 (2H, s), 3.71 (2H, q),
2.70 (2H, t), 1.33 (9H, s).
1
) 87-90 °C. Anal. (C30H38F3N3O5) C, H, N. H NMR (DMSO-
d6), 400 MHz: δ 8.60 (s, 1H), 8.48 (t, 1H), 7.75 (d, 2H), 7.52 (d,
2H), 7.28 (d, 2H), 7.21 (d, 2H), 4.70 (s, 2H), 4.30-4.20 (m, 1H),
3.60 (s, 3H), 3.48 (dd, 2H), 2.55 (t, 2H), 1.82-1.70 (m, 2H), 1.60-
1.45 (m, 4H), 1.40-1.10 (m, 3H), 0.80 (s, 9H).
cis-3-{4-[1-(4-tert-Butylcyclohexyl)-3-(4-trifluoromethoxyphenyl)-
ureidomethyl]benzoylamino}propionic acid ethyl ester (0.2 g, 0.3
mmol) was suspended in EtOH (8 mL), and NaOH (4 N, 0.6 mL)
was added. The reaction mixture was stirred for 16 h at 20 °C and
then concentrated in vacuo until all EtOH was removed. The
reaction mixture was diluted with water (50 mL) and adjusted to
pH 2 with hydrochloric acid (4 N). The aqueous phase was extracted
with EtOAc (80 mL), and the organic phase was dried (MgSO4)
and concentrated in vacuo to give cis-3-{4-[1-(4-tert-butylcyclo-
hexyl)-3-(4-trifluoromethoxyphenyl)ureidomethyl]benzoylamino}-
Supporting Information Available: Details of biological
methods that were used and elemental analysis results. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
1
propionic acid 26. Anal. (C29H36F3N3O5‚0.25H2O) C, H, N. H
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4-Formylbenzoic acid (15 g, 100 mmol) was dissolved in DMF
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128 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
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